HPLC测定大鼠海马中谷氨酸、γ-氨基丁酸的含量

Study on the Determination of Glutamic Acid and γ-Aminobutyric Acid in Hippocampus of Rat by HPLC

关键词:  谷氨酸  γ-氨基丁酸  邻苯二甲醛  高效液相色谱法  大鼠海马

Key words:  glutamic acid  γ-aminobutyric acid  O-phthalaldehyde  HPLC  hippocampus of rat

摘要:

目的 建立了邻苯二甲醛(OPA)柱前衍生高效液相荧光色谱法检测大鼠海马中谷氨酸(GLU)、γ-氨基丁酸(GABA)含量的方法。方法 采用Alltima C18色谱柱(4.6 mm×250 mm，5 μm)，流动相为50 mmol·L-1乙酸钠(pH 6.8)、甲醇、四氢呋喃梯度洗脱，流速1.0 mL·min-1。激发波长(λEx)338 nm，发射波长(λEm)425 nm。结果 GLU、GABA线性范围分别为1.03～20.6 μg·mL-1(r=0.999 2)和1.13～22.6 μg·mL-1(r=0.999 7)；当信噪比为3时，GLU和GABA的检出限分别为5.27×10-4μg·mL-1和7.35×10-4 μg·mL-1；测得精密度RSD分别为3.4%和3.5%；加样回收率分别为90%～96%，85%～91%。结论 本方法简便、准确、灵敏，特异性强，重复性好，适用于海马中GLU和GABA的含量测定。

Abstract:

OBJECTIVE To establish a method for the content of glutamic acid and γ-aminobutyric acid in hippocampus of rat by O-phthalaldehyde(OPA) pre-column derivatization high performance liquid chromatograph-fluorescence (HPLC-FLU). METHODS The separation and determination were carried out by linear gradient elution under following conditions: Alltima C18(250 mm 4.6 mm, 5 μm)column, 50 mmol·L-1acetic acid solution (pH 6.8), methanol and trifluorofuran as mobile phase, flow-rate was 1.0 mL·min-1. The fluorescence excitation and emission wavelengths were set at λEx 338 and λEm 425 nm, respectively. RESULTS The linear range of the method was 1.03-20.6 μg·mL-1(r=0.999 2) for glutamic acid(Glu) and 1.13-22.6 μg·mL-1(r=0.999 7) for r-aminobutyric acid(GABA). The detection limits of GLU and GABA were 5.27×10-4μg·mL-1，and 7.35×10-4μg·mL-1, respectively, when the ratio of signal to noise was 3. The inter-assay precisions were 3.4% and 3.5%, respectively. The recoveries were in the range of 90%-96% and 85%-91%. CONCLUSION The method is simple, sensitive, reliable and has strong specificity and good repetition. It is suitable for the content determination of GLU, GABA in hippocampus of rat by HPLC.